Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A

Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.

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Sample size
Sample size is indicated in the figure legend for each experiments. In vivo experiments, we used mouse numbers that are sufficient for power calculation. The level of significance was set at 5%, and the power was set at 80%.

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Replication Each in vivo experiment were performed with at least n = 4 mice per group. Presented experiments were repeated for a minimum of three times independently with reproducible results. All attempts at replication were successful Randomization Mice used in the present study were randomly assigned to each group.

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nature portfolio | reporting summary Il17atm1.1(icre)Stck (stock number JAX strain #016879) mice, referred as "IL-17AKO", were purchased from Jackson Laboratory (Maine, US) and generated as previously described 60. In these animals the endogenous Il17a gene has been substituted with a Cre-recombinase gene insert inducing total IL-17A deficiency in homozygous mice. MC reconstitution was performed as previously described 25. Briefly, bone-marrow derived MCs (BMMCs) were derived from WT or Il17atm1.1(icre)Stck mice. Bone marrow cells were isolated from the femur of animals. After 3 weeks of differentiation in presence of 10 ng/ml of interleukin 3, mature BMMCs, validated by flow cytometry for high expression of Fcε RI and CD117, were injected i.v.
(106) and locally in the scrotal (i.s.) cavity (106). Four months post-engraftment, mice subjected to TNF-stimulation were analysed for neutrophil infiltration, ICAM-1 and CXCL1 expression. No tissue-infiltrated neutrophils were observed in mice injected with BMMCs at steady state. BMMCs from WT or Il17atm1.1(icre)Stck mice showed similar level of purity (~98%) and expression level of Fcε RI and CD117. All animals were group housed in individually ventilated cages (maximum of 5 mice per cage) under specific pathogen-free (SPF) conditions and a 12-hour (h) light-dark cycle. Room temperature and humidity were maintained within 18-20°C and 30-70% humidity. Food and water were provided ad libitum. At the end of the experiments, mice were euthanised using cervical dislocation. Note that full information on the approval of the study protocol must also be provided in the manuscript.

March 2021
Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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A numerical value for number of cells or percentage (with statistics) is provided.

Sample preparation
Whole blood was collected through the hepatic vein in PBS + 50 mM EDTA. Indicated organs were harvested, mechanically dissociated and treated with 625 U/mL Collagenase I (ThermoFisher) and 100 U/ml DNAse I (Sigma-Aldrich) for 30min at 37°C . Where required, samples were treated with ACK buffer (150 mM NH3Cl, 1 mM KHCO3 and 1 mM EDTA) to lyse red blood cells. Subsequently, single cell suspensions were incubated with anti-CD16/-CD32 antibodies (Becton Dickinson) to block Fcreceptors and stained with primary fluorescently labelled antibodies of interest. Dead cells were excluded using Zombie Aqua Cell population abundance Mast cells, endothelial cells, pericytes abondance were dependant of the organ analysed. In vitro pericyte purity was ~95% post sorting.

Gating strategy
Gating strategy are indicated in supplementary S3, S5 and S6. All gating strategy followed the same pattern: singlets (FSC-A/ FSC-H), debris exclusion, removal of dead cells (negativity for viability marker) Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.